Global stabilization of rRNA structure by ribosomal proteins S4, S17, and S20

Abstract

Ribosomal proteins stabilize the folded structure of the ribosomal RNA and enable the recruitment of further proteins to the complex. Quantitative hydroxyl radical footprinting was used to measure the extent to which three different primary assembly proteins, S4, S17, and S20, stabilize the three-dimensional structure of the Escherichia coli 16S 5' domain. The stability of the complexes was perturbed by varying the concentration of MgCl(2). Each protein influences the stability of the ribosomal RNA tertiary interactions beyond its immediate binding site. S4 and S17 stabilize the entire 5' domain, while S20 has a more local effect. Multistage folding of individual helices within the 5' domain shows that each protein stabilizes a different ensemble of structural intermediates that include nonnative interactions at low Mg(2+) concentration. We propose that the combined interactions of S4, S17, and S20 with different helical junctions bias the free-energy landscape toward a few RNA conformations that are competent to add the secondary assembly protein S16 in the next step of assembly.

Figure 1. Protein interactions in the E. coli 5′ domain

(a) Secondary structure of the 16S rRNA with 5′ domains nucleotides 21-562 in black. Schematic of protein interactions is redrawn from Ref. 12. (b) Structure of the 5′ domain in the E. coli 30S ribosome (2avy; 60), with S4 in pink, S17, green, S20, yellow. Ribbon made with nuccyl (L. Jovine) and Pymol (Delano Scientific).

Figure 2. Mg²⁺-dependent binding of protein S17

Protein S17 was incubated with 1 nM 5′ domain at 37°C in 0-30 mM MgCl₂ (see Methods). (a) Fraction RNA retained on nitrocellulose filters versus S17 concentration. Circles, 30 mM MgCl₂; triangles, no MgCl₂. Symbols and error bars represent the mean and standard deviation of three trials. (b) Equilbrium dissociation constants in 3-30 mM Mg²⁺ concentration. Less than 20% RNA was bound in 0 and 2 mM MgCl₂.

Figure 3. Fe(II)-EDTA footprinting of single protein complexes

Complexes with the 16S 5′ domain were formed at 37°C in 0-35 mM MgCl₂ before cleavage with hydroxyl radical (see Methods). (a) Representative data with S17. Lanes N, no treatment; H, RNA in 80 mM K-Hepes; K, lane H plus 330 mM KCl; A, G, sequence ladders; 30S, native 30S ribosomes. Extension with primer annealing at nt 433 is shown. Protections explained by predicted RNA-RNA contacts in the 30S subunit are shown. (b-d) Fractional saturation of backbone protection (Y) versus Mg²⁺ concentration, relative to RNA in Hepes (Y = 0) and native 30S ribosomes (Y = 1). The data were fit to two or four-state models (Methods). Black, RNA only; pink, +S4, green, +S17; yellow, +S20. (b) nt 288-290 (helix 11); (c) nt 481-483 (helix 17); (d) nt 501-502 (helix 18).

Figure 4. Protein-dependent perturbation of rRNA tertiary interactions

The midpoints for Mg²⁺-dependent saturation of individual contacts along the RNA backbone were sorted into 0.3 mM bins. For residues with two transitions, the highest midpoint was used. Individual parameters are listed in Table S1. Black, RNA only; pink, S4; green, S17; yellow, S20; purple, S4+S17+S20; blue, S4+S16+S17+S20. Data for three and four protein complexes are described elsewhere .

Figure 5. Stabilization of rRNA tertiary structure by ribosomal proteins

Protected regions are colored according to the midpoint (C_m) of the folding transition: red, 0-2.3 mM; orange, 2.3-4.9 mM; green, 4.9-13.4 mM; blue, >13 mM. Residues having more than one transition are clustered according to the one at highest Mg²⁺. 2D schematics and 3D ribbons as in Figure 1. See Supplemental Figure S1 and Table S1 for further data. (a) 5′ domain RNA only; (b) RNA plus S4; (c) RNA plus S17; (d) RNA plus S20. Data in (a) are from .

Figure 6. Primary binding proteins stabilize different rRNA intermediates

In low Mg²⁺, the 5′ domain forms an ensemble of partly folded states (I_C) with a subset of stable tertiary interactions. Individual proteins stabilize partly folded RNAs that may contain non-native interactions. Helices are shown as cylinders (red, most stable; gold, medium stable; green, less stable; blue, disordered). (a) Schematic showing location of helices in the 5′ domain secondary structure; (b) S4; (c) S17; (d) S20.